U.S. patent number 4,248,968 [Application Number 06/024,832] was granted by the patent office on 1981-02-03 for process for producing acrylamide or methacrylamide utilizing microorganisms.
This patent grant is currently assigned to Nitto Chemical Industry Co., Ltd.. Invention is credited to Yoshiaki Satoh, Takayuki Takano, Ichiro Watanabe.
United States Patent |
4,248,968 |
Watanabe , et al. |
February 3, 1981 |
Process for producing acrylamide or methacrylamide utilizing
microorganisms
Abstract
The present invention relates to a process for producing
acrylamide or methacrylamide utilizing microorganisms having a
nitrilase activity. This process involves (1) utilizing highly
active novel bacteria belonging to the genus Corynebacterium or the
genus Nocardia, (2) conducting the reaction utilizing
microorganisms having a nitrilase activity at temperatures as low
as the freezing point of the medium to 15.degree. C. so as to
conduct the reaction for a long period of time while maintaining a
high concentration of acrylamide or methacrylamide, and (3)
conducting the reaction according to a newly devised continuous
column process to obtain a highly concentrated acrylamide or
methacrylamide aqueous solution with economic advantages.
Inventors: |
Watanabe; Ichiro (Yokohama,
JP), Satoh; Yoshiaki (Yokohama, JP),
Takano; Takayuki (Yokohama, JP) |
Assignee: |
Nitto Chemical Industry Co.,
Ltd. (Tokyo, JP)
|
Family
ID: |
27288726 |
Appl.
No.: |
06/024,832 |
Filed: |
March 28, 1979 |
Foreign Application Priority Data
|
|
|
|
|
Mar 29, 1978 [JP] |
|
|
53-35318 |
Apr 28, 1978 [JP] |
|
|
53-51236 |
Apr 28, 1978 [JP] |
|
|
53-51237 |
|
Current U.S.
Class: |
435/129; 435/182;
435/227; 435/293.1; 435/294.1; 435/299.1; 435/813; 435/822;
435/832; 435/840; 435/843; 435/859; 435/872 |
Current CPC
Class: |
C12P
13/02 (20130101); Y10S 435/832 (20130101); Y10S
435/84 (20130101); Y10S 435/859 (20130101); Y10S
435/822 (20130101); Y10S 435/813 (20130101); Y10S
435/872 (20130101); Y10S 435/843 (20130101) |
Current International
Class: |
C12P
13/02 (20060101); C12P 13/00 (20060101); C12P
013/02 () |
Field of
Search: |
;435/129,227,288,813 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4001081 |
January 1977 |
Commeyras et al. |
|
Primary Examiner: Shapiro; Lionel M.
Attorney, Agent or Firm: Sughrue, Rothwell, Mion, Zinn and
Macpeak
Claims
What is claimed is:
1. A process for producing acrylamide or methacrylamide utilizing
microorganisms, which comprises subjecting acrylonitrile or
methacrylonitrile in an aqueous medium to the action of bacteria
belonging to the genus Corynebacterium or the genus Nocardia having
the ability to hydrolyze acrylonitrile or methacrylonitrile, at a
temperature of from the freezing point of the medium to 30.degree.
C. at a pH of 6 to 10.
2. A process for producing acrylamide or methacrylamide utilizing
microorganisms, which comprises subjecting acrylonitrile or
methacrylonitrile in an aqueous medium to the action of bacteria
having the ability to hydrolyze acrylonitrile or methacrylonitrile
at a temperature of 15.degree. C. or less, at a temperature of from
the freezing point of the medium to 15.degree. C. at a pH of 6 to
10.
3. The process of claim 2, wherein said microorganisms are selected
from bacteria belonging to the genus Corynebacterium, the genus
Nocardia, the genus Bacillus, the genus Bacteridium in the sense of
Prevot, the genus Micrococcus and the genus Brevibacterium in the
sense of Bergey.
4. The process of claim 3, wherein said bacteria are from the genus
Corynebacterium or the genus Nocardia.
5. The process of claim 1 or 3, wherein said bacteria are of the
genus Corynebacterium.
6. The process of claim 1 or 3, wherein said bacteria are of the
genus Norcardia.
7. The process of claim 1 or 2, wherein said bacteria are
immobilized with a polymer gel.
8. The process of claim 7, wherein said immobilized bacteria are
entrapped with a polyacrylamide and related polymer gel.
9. A process for continuously producing a highly concentrated
acrylamide or methacrylamide aqueous solution which comprises
passing an aqueous solution of acrylonitrile or methacrylonitrile
through one or more columns filled with immobilized bacterial cells
having a nitrilase activity at a temperature of from the freezing
point of the medium to 30.degree. C. at a pH of 6 to 10 while
feeding acrylonitrile or methacrylonitrile via one or more inlets
intermediate the column inlet and outlet in an amount soluble in
the reaction mixture.
10. The process of claim 9, wherein at least two columns connected
in series are used and an aqueous solution of acrylonitrile or
methacrylonitrile is fed to the first column inlet while
acrylonitrile or methacrylonitrile are fed via the second and
subsequent column inlets.
11. The process of claim 9 or 10, wherein said bacteria are
selected from the group consisting of the genus Corynebacterium,
the genus Nocardia, the genus Bacillus, the genus Bacteridium in
the sense of Prevot, the genus Micrococcus and the genus
Brevibacterium in the sense of Bergey.
12. The process of claim 9 or 10, wherein said bacteria are
immobilized with a polymer gel.
13. The process of claim 12, wherein said immobilized bacteria are
entrapped with a polyacrylamide and related polymer gel.
14. The process of claim 9 or 10, wherein the reaction is conducted
at a temperature of from the freezing point of the medium to
15.degree. C.
15. The process of claim 11, wherein said bacteria are from the
genus Corynebacterium or the genus Nocardia.
16. The process of claim 15, wherein said bacteria are of the genus
Corynebacterium.
17. The process of claim 15, wherein said bacteria are of the genus
Nocardia.
18. The process of claim 1, 2, 9 or 10, wherein said bacteria are
the strains N-771 or N-774 belonging to the genus Corynebacterium
or the strain N-775 belonging to the genus Nocardia.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an improved process for producing
acrylamide or methacrylamide utilizing microorganisms.
2. Description of the Prior Art
As a process for producing acrylamide or methacrylamide, there has
heretofore been known a process of reacting acrylonitrile (AN) or
methacrylonitrile (MAN) with water using reduced copper as a
catalyst. However, it has been desired to develop a novel and
industrially more advantageous process since the catalytic process
involves a difficult catalyst preparation and regeneration, and the
isolation and purification of the amide produced is onerous.
On the other hand, as a process for producing acrylamide or
methacrylamide from acrylonitrile or methacrylonitrile utilizing an
enzymatic reaction, an interesting process using bacteria belonging
to the genus Bacillus, the genus Bacteridium in the sense of
Prevot, the genus Micrococcus, the genus of Brevibacterium in the
sense of Bergy, or the like has recently been proposed in U.S. Pat.
No. 4,001,081. This process is merely based on the discovery that
the above-described bacteria hydrolyze various organic nitriles to
produce the corresponding organic acid amides. In the case of using
acrylonitrile or methacrylonitrile (Examples 6-8 in the Patent) for
example, the patent describes that acrylamide or methacrylamide was
obtained almost quantitatively under the reaction conditions of: 8
to 12 wt % acrylonitrile or methacrylonitrile concentration, 2 to 4
wt % bacterial cell concentration, 7 to 9 in pH, 25.degree. C. in
temperature and 20 to 30 minutes reaction time. It is true that
acrylamide or methacrylamide can be produced at a concentration as
high as 10 to 20 wt %, but the bacterial cells so rapidly lose
their enzymatic activity under such conditions that it is almost
impossible to use them repeatedly. In addition, the solution from
which the bacterial cells are separated is colored an extremely
dark yellow and contains various impurities originating from the
cells, and hence an onerous purifying step is necessary. Thus, the
abovedescribed process is not economically advantageous in
industrial applications.
SUMMARY OF THE INVENTION
A novel catalytic process for producing acrylamide or
methacrylamide utilizing microorganisms has been investigated and
bacteria having an extremely high activity for hydrolyzing
acrylonitrile and methacrylonitrile to produce acrylamide or
methacrylamide have been discovered. Namely the strain N-771 and
the strain N-774 belonging to the genus Corynebacterium, and the
strain N-775 belonging to the genus Nocardia have been found in the
soils around the factory producing acrylonitrile and in the waste
water discharged from the factory. (Hereafter the aforementioned
bacteria will be referred to as N-771, N-774 and N-775,
respectively.) The enzymatic nitrilase activity of these
microorganisms is surprisingly high at low temperatures. As a
result of intensive investigations, a process for the hydrolysis of
acrylonitrile and methacrylonitrile has been developed wherein the
enzymatic activity of the bacterial cells is stably maintained at a
high level for a long time, with the accumulation of produced
acrylamide or methacrylamide reaching concentrations as high as 10
wt % or more, which process does not require a difficult purifying
step.
Thus, a principal object of the present invention is to provide a
process for producing acrylamide or methacrylamide utilizing
microorganisms, which comprises subjecting acrylonitrile or
methacrylonitrile in an aqueous medium to microorganisms belonging
to the genus Corynebacterium or the genus Nocardia and having the
ability of hydrolyzing acrylonitrile or methacrylonitrile, at a
temperature ranging from the freezing point of the medium to
30.degree. C. at a pH of about 6 to 10.
Another object of the present invention is to provide a process for
producing acrylamide or methacrylamide utilizing microorganisms,
which comprises subjecting acrylonitrile or methacrylonitrile in an
aqueous medium to microorganisms having the ability to hydrolyze
acrylonitrile or methacrylonitrile to produce acrylamide or
methacrylamide, at a temperature ranging from the freezing point of
the medium to 15.degree. C. at a pH of about 6 to 10.
A further object of the present invention is to provide a process
for continuously producing a highly concentrated acrylamide or
methacrylamide aqueous solution by passing an aqueous solution of
acrylonitrile or methacrylonitrile through a column or columns
filled with immobilized bacterial cells having nitrilase activity,
at a temperature ranging from the freezing point of the solution to
30.degree. C. at a pH of about 6 to 10, which comprises:
(1) using a column having one or more feed inlets provided between
the column inlet and the column outlet, continuously feeding an
aqueous solution of acrylonitrile or methacrylonitrile via said
column inlet and, at the same time, continuously feeding
acrylonitrile or methacrylonitrile via said feeding inlet(s) an
amount soluble in the reaction medium; or
(2) using two or a plurality of columns connected to each other in
series, and continuously feeding an aqueous solution of
acrylonitrile or methacrylonitrile via the first column inlet and,
at the same time, continuously feeding acrylonitrile or
methacrylonitrile via the column inlet(s) of the successive columns
in an amount soluble in the reaction mixture.
DETAILED DESCRIPTION OF THE INVENTION
As the microorganisms used in the present invention, any one that
has the ability to hydrolyze acrylonitrile or methacrylonitrile to
produce acrylamide or methacrylamide may be used regardless of the
taxonomic position, as well as the aforesaid strains N-771, N-774
and N-775. For example bacteria from the genus Bacillus, the genus
Bacteridium, the genus Micrococcus and the genus Brevibacterium as
disclosed in U.S. Pat. No. 4,001,081 may also be used. In addition,
it is also possible to use the cellular extract prepared by
destroying such bacterial cells, crude enzyme preparations,
etc.
To culture the microorganisms used in the present invention,
ordinary culture mediums containing a carbon source (e.g., glucose,
maltose, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium
chloride, etc.), an organic nutrient source (e.g., yeast extract,
malt extract, peptone, meat extract, etc.), and an inorganic
nutrient source (e.g., phosphate, magnesium, potassium, zinc, iron,
manganese, etc.) are used. The culture is aerobically conducted
while maintaining the pH of the culture medium at about 6 to 9 at a
temperature of about 20.degree. to 35.degree. C., preferably about
25.degree. to 30.degree. C., for about 1 to 5 days.
The strains N-771, N-774 and N-775 to be used in the present
invention are deposited at Fermentation Research Institute, Agency
of Industrial Science & Technology, Ministry of International
Trade and Industry, Japan, as FERM-P Nos. 4445, 4446 and 4447,
respectively. The bacteriological characteristics of each strain
are as shown below.
A: STRAIN N-771
(a) Morphology
(1) Shape and size of cells: (0.5-0.8).mu..times.(2-5).mu.
(2) Pleomorphism of cells: At the initial stage of culture, the
bacterial cells are in a long bacillary form of rods without
bending, and grow with snapping and, later, break and split into a
coccoid or short bacillary form.
(3) Motility: none
(4) Spore: none
(5) Gram straining: positive
(6) Acid fastness: negative
(7) Metachromatic granules: positive
(b) Growth state in various culture mediums (at 30.degree. C.)
(1) Nutrient agar plate culture: Circular (1-3 mm in diameter),
with solid edges, smooth, hemispherical, opaque with luster,
slightly pink.
(2) Nutrient agar slant culture: Middle growth, filament-like,
surface-smooth, convex, with luster, slightly pink.
(3) Bouillon liquid culture: Vigorous growth with forming pellicle,
middle-degree turbidity with growth, forming a precipitate.
(4) Bouillon gelatin stab culture: Good growth on the surface,
funnel-like growth along stab, with almost no growth at the lower
portion, no liquefaction of gelatin.
(5) Litmus milk: no change
(c) Physiological characteristics
(1) Reduction of nitrate: positive
(2) Dentrification: negative
(3) MR test: negative
(4) VP test: negative
(5) Indole production: negative
(6) Hydrogen sulfide production: negative
(7) Hydrolysis of starch: negative
(8) Citric acid use:
Koser's culture medium: negative
Christiansen's culture medium: positive
(9) Use of inorganic nitrogen source:
Nitrate: positive
Ammonium salt: positive
(10) Pigment production: negative
(11) Urease: positive
(12) Oxidase: negative
(13) Catalase: positive
(14) Hydrolysis of cellulose: negative
(15) Growth range: pH: 5-10; temp.: 5.degree.-37.degree. C.
(16) Oxygen relation: aerobic
(17) O-F test: F
(18) Heat resistance (in 10% skim milk, at 72.degree. C. for 15
minutes): none
(19) Acid and gas production from sugar
______________________________________ Acid production Gas
production ______________________________________ L-Arabinose + -
D-Xylose - - D-Glucose + - D-Mannose - - D-Fructose + - D-Galactose
- - Maltose - - Sucrose - - Lactose - - Trehalose - - D-Sorbitol +
- D-Mannitol + - Inositol - - Glycerin + - Starch - - Salicin - -
______________________________________
B: STRAIN N-774
(a) Morphology
(1) Shape and size of cells: (0.5-0.8).mu..times.(2-5).mu.
(2) Pleomorphism of cells: At the initial stage of culture, the
bacterial cells are in a long bacillary form of rods without
bending, and grow with snapping and, later, break and split into a
coccoid or short bacillary form.
(3) Motility: none
(4) Spore: none
(5) Gram straining: positive
(6) Acid fastness: negative
(7) Metachromatic granules: positive
(b) Growth state in various culture mediums (at 30.degree. C.)
(1) Nutrient agar plate culture: Circular (1-3 mm in diameter),
slightly irregular, smooth with surface-drying tendency, flat,
opaque, slightly pink.
(2) Nutrient agar slant culture: Middle growth, filament-like,
surface-smooth, convex with drying tendency, slightly pink.
(3) Bouillon liquid culture: Vigorous growth with forming pellicle,
slight turbidity, forming a precipitate with growth.
(4) Bouillon gelatin stab culture: Good growth on the surface,
funnel-like growth along stab, with almost no growth at the lower
stab portion, no liquefaction of gelatin.
(5) Litmus milk: no change
(c) Physiological characteristics
(1) Reduction of nitrate: positive
(2) Dentrification: negative
(3) MR test: negative
(4) VP test: negative
(5) Indole production: negative
(6) Hydrogen sulfide production: negative
(7) Hydrolysis of starch: negative
(8) Citric acid use:
Koser's culture medium: negative
Christiansen's culture medium: positive
(9) Use of inorganic nitrogen source:
Nitrate: positive
Ammonium salt: positive
(10) Pigment production: negative
(11) Urease: positive
(12) Oxidase: negative
(13) Catalase: positive
(14) Hydrolysis of cellulose: negative
(15) Growth range: pH: 5-10; temp.: 10.degree.-40.degree. C.
(16) Oxygen relation: aerobic
(17) O-F test: F
(18) Heat resistance (in 10% skim milk, at 72.degree. C. for 15
minutes): none
(19) Acid and gas production from sugar
______________________________________ Acid production Gas
production ______________________________________ L-Arabinose + -
D-Xylose - - D-Glucose + - D-Mannose + - D-Fructose + - D-Galactose
- - Maltose + - Sucrose - - Lactose - - Trehalose .+-. - D-Sorbitol
+ - D-Mannitol + - Inositol - - Glycerin + - Starch - - Salicin
.+-. - ______________________________________
C: STRAIN N-775
(a) Morphology
(1) Shape and size of cells: (0.6-1.0).mu..times.(5-15).mu.
(2) Pleomorphism of cells: At the initial stage of culture, the
bacterial cells are in a long bacillary form with hyphalike
appearance, and grow with branching and, later, break and split
into a coccoid or short bacillary form.
(3) Motility: none
(4) Spore: none
(5) Gram staining: positive
(6) Acid fastness: weakly positive
(7) Metachromatic granules: positive
(b) Growth state in various culture mediums (at 30.degree. C.)
(1) Nutrient agar plate culture: Circular (1-3 mm in diameter),
irregular, smooth, in relief, opaque, slightly lustrous, slightly
red.
(2) Nutrient agar slant culture: Middle growth, filament-like,
surface-smooth, flat trapezoid cross section with slight luster,
slightly red.
(3) Bouillon liquid culture: Vigorous growth with forming pellicle,
transparent solution, slightly forming a precipitate with
growth.
(4) Bouillon gelatin stab culture: Good growth on the surface,
funnel-like growth along stab, with almost no growth at the lower
stab portion, no liquefaction of gelatin.
(5) Litmus milk: no change
(c) Physiological characteristics
(1) Reduction of nitrate: positive
(2) Dentrification: negative
(3) MR test: negative
(4) VP test: negative
(5) Indole production: negative
(6) Production of hydrogen sulfide: negative
(7) Hydrolysis of starch: negative
(8) Citric acid use:
Koser's culture medium: positive
Christiansen's culture medium: positive
(9) Use of inorganic nitrogen source:
Ammonium salt: positive
Nitrate: positive
(10) Pigment production: negative
(11) Urease: positive
(12) Oxidase: negative
(13) Catalase: positive
(14) Hydrolysis of cellulose: negative
(15) Growth range: pH: 6-10; temp.: 10.degree.-40.degree. C.
(16) Oxygen relation: aerobic
(17) O-F test: 0
(18) Heat resistance (in 10% skim milk, at 72.degree. C. for 15
minutes): none
(19) Acid and gas production from sugar:
______________________________________ Acid production Gas
production ______________________________________ D-Arabinose + -
D-Xylose + - D-Glucose + - D-Mannose - - D-Fructose + - D-Galactose
+ - Maltose - - Sucrose + - Lactose - - Trehalose .+-. - D-Sorbitol
+ - D-Mannitol + - Inositol - - Glycerin + - Starch - - Salicin - -
______________________________________
To determine taxonomic positions of the bacteria based on the
above-described bacteriological characteristics according to
Bergy's Manual of Determinative Bacteriology, 7th ed. (1957) and
8th ed. (1974), the strains N-771 and N-774 fall under the aerobic,
Gram-positive, non-acid fastness and catalase-positive bacillary
category forming no endo-spores and no flagella. From the fact that
the bacteria are in a long bacillary form at the initial stage of
growth, not showing filament-like appearance but showing snapping
growth without branching and that the bacteria break and split into
a coccoid or short bacillary form, it is clear that they fall under
the category of Coryneform bacteria. In addition, comparison with
the Coryneform bacteria described in the Bergy's Manual precludes
the bacteria of the present invention from belonging to: (1) the
genus Cellulomonas, because they do not have cellulose-decomposing
ability, (2) the genus Arthrobacter, because Gram-staining is not
variable, (3) the genus Microbacterium, because they do not have
heat resistance in 10% skim milk at 72.degree. C. for 15 minutes,
and (4) the genus Kurthia because they do not have flagella.
Accordingly, it is concluded the bacteria of the present invention
belong to the genus Corynebacterium.
The strain N-775 falls under the aerobic, Gram-positive, weakly
acid fastness and catalase-positive bacillary category forming no
endospores and no flagella. From the fact that the bacteria are in
a long bacillary form at the initial stage of growth, showing
hypha-like appearance, and grow with branching to break and split
later into a coccoid or short bacillary form, they are considered
to belong to the genus Nocardia.
In practicing the process of the present invention, all that is
required is to select a microorganism having the ability to
hydrolyze acrylonitrile or methacrylonitrile or one of the
above-described microorganisms, culture it for 2 to 3 days in the
aforesaid manner, collect the bacterial cells from the culture
solution by centrifugation, suspend the cells in water or
physiological saline and subject acrylonitrile or methacrylonitrile
to the action of the cells.
That is, the reaction may usually be conducted in an aqueous
suspension containing about 1 to 10 dry wt % of bacterial cells and
0.5 to 10 wt % of acrylonitrile or methacrylonitrile at a
temperature ranging from about the freezing point of the medium to
30.degree. C., preferably from about the freezing point to
15.degree. C., at a pH of about 6 to 10, preferably about 7 to 9,
for about 0.5 to 10 hours. Additionally, upon reaction, it is
preferable to subsequently add acrylonitrile or methacrylonitrile
as their concentration in the system falls while limiting the
concentration of acrylonitrile or methacrylonitrile in the system
to a level of not higher than 2 wt %, since they possess a strong
toxicity and would inhibit the enzymatic reaction. Generally
slightly higher concentrations of acrylonitrile and
methacrylonitrile are possible with the batch process than with the
continuous process described below because it is possible to stir
the reaction system and a homogeneous system can be obtained.
During the reaction, the pH is preferably controlled to be in the
range of about 7 to 9 by consecutively adding a caustic alkali,
ammonia or the like or by previously adding a buffer solution to
the system. pH values outside the above range would lead to further
hydrolysis of the produced and accumulated acrylamide or
methacrylamide to form by-products or would lead to reduction in
stability of the cell enzyme. Thus, acrylamide or methacrylamide
can be produced and accumulated with almost 100% conversion.
It is particularly noted that the accumulated concentration of
produced acrylamide or methacrylamide attainable and the life of
cell enzyme activity are remarkably improved by conducting the
reaction at a temperature as low as the freezing point of the
medium to 15.degree. C., which is based on the following knowledge
which has so far been unexpected.
That is, it has been found that: (1) the nitrilase as the hydrolase
of the present invention produced and accumulated in the aforesaid
bacterial cells has an extremely higher activity than generally
well known hydrolases by 10 to 50 times and, therefore, the
reaction can be conducted at an economical reaction rate even at
temperatures as low as 15.degree. C. or lower, (2) the enzyme of
the present invention has relatively low heat resistance and it
could be inactivated in an extremely short time at temperatures
usually employed for ordinary enzymatic reactions (25.degree. to
30.degree. C.), and hence its effect is not fully exhibited when
various chemical treatments are conducted to assure its stability,
unless the reaction is conducted at low temperatures, (3) since the
enzyme of the present invention has a relatively high activity and
can react at low temperature, it is possible to remarkably reduce
the enzymatic activity inhibition of acrylonitrile or
methacrylonitrile and acrylamide or methacrylamide, and as a result
attain concentrations of accumulatd acrylamide or methacrylamide as
high as 10 to 30 wt % while stably maintaining the enzymatic
activity for a long period of time.
These microorganisms may be used as intact cells but, from the
standpoint of repeated use, continuous operation and purification,
immobilized cells, in particular, immobilized cells entrapped by a
polyacrylamide and related polymer gels, are preferred.
In conventional immobilized cells prepared by entrapping cells with
polyacrylamide and related polymer, the level of enzymatic activity
in the immobilized cells is 30 to 60% of the activity of intact
cells in most cases. On the other hand, the cells of the present
invention can be immobilized at the activity level of almost 100%,
because the microorganisms are acrylamide-producing bacteria and
are stable against highly concentrated acrylamide, and because
immobilizing can be conducted at 15.degree. C. or less.
The cell immobilization can be conducted by suspending the
aforesaid microorganisms in a suitable aqueous medium (e.g., water,
a physiological saline, a buffer solution, etc.) containing an
acrylamide series monomer and a cross linking agent, adding a
suitable polymerization initiator and a polymerization accelerator
to the suspension, and conducting polymerization and gellation at
about 0.degree. to 30.degree. C., preferably 0.degree. to
15.degree. C., at a pH of about 5 to 10, preferably about 6 to 8.
The content of microorganisms in the polymerization reaction
solution depends upon the kind and the form of the microorganisms
used, but it is usually about 0.1 to 50 wt %, preferably about 1 to
20 dry wt %.
The acrylamide series monomers used to immobilize the cells in the
present invention include, for example, acrylamide, methacrylamide,
etc. and, if necessary, ethylenically unsaturated monomers
copolymerizable with them may be used in combination. The
concentration of such monomers in the reaction should at least be
at a level high enough to form gels as a result of the
polymerization, and is usually about 2 to 30 wt %, preferably about
5 to 20 wt %, based on the reaction solution.
The cross-linking agents include N,N'-methylenebisacrylamide,
1,3-di-(acrylamidomethyl)-2-imidazolidone, etc. As the
polymerization initiator and the polymerization accelerator, those
which least inhibit the activity of microorganisms are selected.
Usually, potassium persulfate, ammonium persulfate, etc. are used
as the initiator, and dimethylaminopropionitrile, triethanolamine,
etc. are used as the accelerator, each in an amount of about 0.01
to 10 wt %.
Thus, there can be obtained polymer gels containing bacterial
cells, i.e., immobilized cells.
The reaction of the present invention may be conducted either in a
batchwise manner or in a continuous manner, but the use of the
above-described immobilized cells and the continuous column process
described hereinafter enables one to obtain a highly concentrated
acrylamide or methacrylamide aqueous solution with extremely good
industrial advantages through relatively simple procedures while
stably maintaining the activity of cell enzyme for a long time.
That is, the continuous column process in accordance with the
present invention comprises using one or a plurality of columns
connected to each other in series, which are filled with the
aforesaid immobilized cells in a density of about 0.3 to 0.5 g
immobilized cells/cc. having been crushed to a suitable size (about
0.5 to 5 mm, preferably about 1 to 3 mm), continuously feeding an
aqueous solution of acrylonitrile or methacrylonitrile via column
inlet and, at the same time, continuously feeding acrylonitrile or
methacrylonitrile at an intermediate stage or location before
completion of the reaction in an amount soluble in the reaction
solution. In more detail, where one column is used, a so-called
sectional column having one or more feed inlets provided between
the column inlet and the column outlet (one feed inlet per
section), and which usually comprises a few sections, is
preferable. An aqueous solution of acrylonitrile or
methacrylonitrile is continuously fed via the column inlet and, at
the same time, acrylonitrile or methacrylonitrile is continuously
fed via all the feed inlets. A suitable feed rate is usually about
0.1 to 1.5 g AN or MAN/g cell.hr., preferably 0.3 to 0.8 g AN or
MAN/g cell.hr. The amount of acrylonitrile or methacrylonitrile fed
via each feed inlet is such that the acrylonitrile or
methacrylonitrile added is soluble in the reaction mixture. It is
preferable that the concentration of acrylonitrile or
methacrylonitrile in the reaction system is limited to a level of
not higher than 2 wt % since as explained above at higher
concentrations it begins to have a toxic effect and inhibit the
enzymatic reaction. Feeding rates in respective inlets are not
necessarily the same due to the difference in the rate of
consumption of acrylonitrile or methacrylonitrile during the
progress of the reaction, i.e., the rates will vary depending on
whether the acrylonitrile or methacrylonitrile is soluble at that
particular level.
Where two or more columns are used, they are connected to each
other in series, and an aqueous solution of acrylonitrile or
methacrylonitrile is fed via the column inlet of the first column,
and acrylonitrile or methacrylonitrile is fed via the subsequent
and successive column inlet(s) in the same manner as described
above. Thus, there can be obtained a highly concentrated acrylamide
or methacrylamide solution as an eluate from the second or final
column.
On the other hand, in the conventional continuous column process,
it has been difficult to bring the bacterial cells into uniform
contact with a material having a low solubility in water such as
acrylonitrile of methacrylonitrile at a high concentration to react
and, therefore, the conventional process suffers from the defects
that a highly concentrated acrylamide or methacrylamide aqueous
solution cannot be obtained effectively and smoothly, and that the
enzymatic activity of the bacterial cells is sharply reduced.
Additionally, a more concentrated acrylamide or methacrylamide
aqueous solution or crystals of acrylamide or methacrylamide can be
obtained from the thus obtained acrylamide or methacrylamide
aqueous solution of the present invention using conventional
techniques. For example, the aforesaid reaction solution is
treated, if necessary, with active carbon, ion-exchange resin,
etc., and then concentrated under reduced pressure to obtain a more
concentrated acrylamide or methacrylamide aqueous solution or
crystals thereof.
The present invention will now be described in more detail by the
following examples of preferred embodiments of the present
invention which, however, should not be construed as limiting the
present invention. Additionally, all parts and percents in the
following examples are by weight. The reaction products such as
acrylamide and methacrylamide, unreacted materials such as
acrylonitrile and methacrylonitrile, and by-products like
methacrylic acid and acrylic acid were determined by means of gas
chromatography.
EXAMPLE 1
12.5 parts of the washed cells of the strain N-771 (water content:
80%) prepared by aerobic culture using a culture medium (pH: 7.2)
containing 1% glucose, 0.5% peptone, 0.3% yeast extract and 0.3%
malt extract, 6 parts of acrylonitrile and 81.5 parts of a 0.05 M
phosphate buffer (pH: 8.8) were mixed and reacted for 1 hour at
30.degree. C. under stirring. After the completion of the reaction,
the cells were removed by centrifugation to obtain a clear
solution. This solution contained 8.0% acrylamide, but contained no
unreacted acrylonitrile and no by-products like acrylic acid. Thus,
the reaction proceeded almost quantitatively to completion.
EXAMPLE 2
12.5 parts of the washed cells of the strain N-771 (water content:
80%) prepared in the same manner as in Example 1 were mixed with
87.5 parts of water, and acrylonitrile was continuously added
dropwise thereto at a rate of 4 parts per hour while controlling
the pH at 8.0 using potassium hydroxide under stirring to react at
30.degree. C. After reacting for 2.5 hours, dropwise addition of
acrylonitrile was stopped, followed by stirring for further 30
minutes. The resulting reaction solution having been reacted for
further 30 minutes was centrifuged to remove the cells and obtain a
clear solution. This solution contained 12.0% acrylamide, but
absolutely no unreacted acrylonitrile was detected. Thus, the
reaction was completed.
EXAMPLE 3
15 parts of the washed cells of the strain N-771 (water content:
80%) prepared in the same manner as in Example 1, 8 parts of
methacrylonitrile and 77 parts of a 0.05 M phosphate buffer (pH:
8.8) were mixed and reacted for 1 hour at 30.degree. C. After
completion of the reaction, the cells were removed by
centrifugation to obtain a clear solution. This solution contained
10.2% methacrylamide. Although a trace of methacrylic acid was
detected, unreacted methacrylonitrile was not detected at all.
Thus, the reaction proceeded almost quantitatively to
completion.
EXAMPLE 4
12 parts of the washed cells of the strain N-774 (water content:
75%) prepared in the same manner as in Example 1 were mixed with 88
parts water, and methacrylonitrile was continuously added dropwise
thereto at a rate of 3 parts per hour while controlling the pH of
the solution at 8.5 using potassium hydroxide under stirring to
react at 30.degree. C. After reacting for 4 hours, dropwise
addition of methacrylonitrile was discontinued, followed by
stirring for further 30 minutes to almost completely react the
methacrylonitrile within the system. After completion of the
reaction, the cells were removed by centrifugation to obtain a
clear solution. Methacrylamide in this solution was determined to
be 13.0%.
EXAMPLE 5
25 parts of the washed cells (water content: 78%) of the strain
N-775 prepared by aerobic culture using a culture medium (pH: 7.2)
containing 1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt
extract, 0.1% acetonitrile, 0.1% KH.sub.2 PO.sub.4 and 0.05%
MgSO.sub.4.7H.sub.2 O, 5 parts of acrylonitrile and 70 parts of a
0.05 M phosphate buffer (pH: 8.8) were mixed and reacted for 1 hour
at 30.degree. C. under stirring. After the completion of the
reaction, the cells were removed by centrifugation to obtain a
clear solution. This solution contained 6.7% acrylamide, but
absolutely no unreacted acrylonitrile and no by-products like
acrylic acid were detected. Thus, the reaction proceeded almost
quantitatively to completion.
EXAMPLE 6
8 parts of the washed cells of the strain N-771 (water content:
75%) prepared by aerobic culture using a culture medium (pH: 7.2)
containing 1% glucose, 0.5% peptone, 0.3% yeast extract and 0.3%
malt extract was mixed with 92 parts water, and acrylonitrile was
intermittently added dropwise at a rate of 2 parts per hour while
controlling the pH of the solution at 8.0 by properly adding a 0.5
N KOH aqueous solution under stirring, at various reaction
temperatures ranging from about 0.degree. C. to 30.degree. C. as
shown in Table 1. The reaction was continued until unreacted
acrylonitrile was detected and, at that stage, the reaction was
stopped and the cells were removed by centrifugation to obtain a
clear solution. The content of acrylamide was determined with
respect to each solution to compare the concentrations of
accumulated acrylamide at respective reaction temperatures. Thus,
the results shown in Table 1 were obtained. It is seen from the
results, that the enzymatic activity of the cells became stable and
the concentration of produced and accumulated acrylamide was
greatly increased when the reaction was conducted at temperatures
of not higher than 15.degree. C.
Table 1 ______________________________________ Run No. 6-1 6-2 6-3
6-4 6-5 6-6 ______________________________________ Reaction
Temperature -3 to 0 5 10 15 20 30 (.degree. C.) Reaction Time
Before Acrylonitrile was 16 16 14 12 5 4 Detected (hrs.) Acrylamide
in the Reaction Solution (%) 31.8 31.0 28.1 25.0 10.7 9.3
______________________________________
EXAMPLE 7
13.5 parts of the washed cells of the strain N-775 (water content:
78%) obtained by culturing in the same manner as in Example 6 were
mixed with 86.5 parts water, and acrylonitrile was intermittently
added dropwise at a rate of 2 parts per hour while controlling the
pH of the solution at 8.0 by properly adding a 0.5 N potassium
hydroxide aqueous solution under stirring, at various reaction
temperatures ranging from about 0.degree. C. to about 30.degree. C.
as shown in Table 2. Subsequently, the reaction was continued in
the same manner as in Example 6 until unreacted acrylonitrile was
detected. The concentration of acrylamide was determined with
respect to each solution to obtain the results in Table 2. It is
seen in this example, too, that the produced and accumulated
acrylamide concentration greatly increased in the experiments
wherein the reaction was conducted at temperature of not higher
than 15.degree. C.
Table 2 ______________________________________ Run No. 7-1 7-2 7-3
7-4 7-5 7-6 ______________________________________ Reaction
Temperature -3 to 0 5 10 15 20 30 (.degree. C.) Reaction Time
Before Acrylonitrile was 14 13 11 10 5 4 Detected (hrs.) Acrylamide
in the Reaction Solution (%) 28.2 27.5 23.1 21.0 11.5 9.5
______________________________________
EXAMPLE 8
13.5 parts of the washed cells of the strain CBS 717.73 (the strain
described in the Examples of U.S. Pat. No. 4,001,081) obtained by
culturing in the same manner as in Example 6 (water content: 78%)
was mixed with 86.5 parts water, and acrylonitrile was
intermittently added dropwise at a rate of 2 parts per hour while
controlling the pH of the solution at 8.0 by properly adding a 0.5
N potassium hydroxide aqueous solution under stirring, at various
reaction temperatures ranging from about 0.degree. C. to 30.degree.
C. as shown in Table 3. Subsequently, the reaction was continued in
the same manner as in Example 6 until unreacted acrylonitrile was
detected. The acrylamide concentration of each solution was
determined. The results obtained are shown in Table 3. It is seen
in this example, too, that the produced and accumulated acrylamide
concentration greatly increased in the experiments wherein the
reaction was conducted at temperatures of not higher than
15.degree. C.
Table 3 ______________________________________ Run No. 8-1 8-2 8-3
8-4 8-5 8-6 ______________________________________ Reaction
Temperature -3 to 0 5 10 15 20 30 (.degree. C.) Reaction Time
Before Acrylonitrile was Detected (hrs.) 13 13 11 10 5 4 Acrylamide
in the Reaction Solution (%) 27.5 26.2 22.9 21.3 10.2 9.5
______________________________________
EXAMPLE 9
4 parts of the washed cells of the strain N-771 obtained in the
same manner as in Example 6, 0.45 parts of acrylamide, 0.05 part of
N,N'-methylenebisacrylamide and 4 parts of physiological saline
were mixed to prepare a uniform suspension. To this suspension were
added 0.5 part of a 5% dimethylaminopropionitrile aqueous solution
and 1 part of a 2.5% potassium persulfate aqueous solution, and the
system was maintained at 10.degree. C. for 30 minutes to
polymerize. The thus obtained massive, cell-containing gels were
crushed into small particles and washed with physiological saline
to obtain 10 parts of immobilized cells. To 20 parts of the
immobilized cells was added 72 parts of a 0.05 M phosphate buffer
(pH: 8.0), and acrylontrile was dropwise added thereto at a rate of
2 parts per hour to react for 4 hours under stirring. A clear
solution obtained by separating and removing the bacterial cells
from the reaction product contained 10.6% acrylamide and almost no
by-products like acrylic acid and unreacted acrylonitrile were
detected. Thus the reaction proceeded almost quantitatively to
completion.
The separated immobilized cells were repeatedly used to conduct the
same reaction. On the other hand, similar experiments were
conducted at 30.degree. C. for comparison. The results obtained are
shown in Table 4.
Table 4 ______________________________________ Number of times of
repeatedly using the Yield of acrylamide (%) microorganisms
15.degree. C. 30.degree. C. ______________________________________
1 100 100 2 100 100 3 100 85 4 100 5 5 100 0 6 100 0 7 100 0
______________________________________
EXAMPLE 10
40 g of the immobilized cells of the strain N-771 obtained in the
same manner as in Example 9 were filled in a jacketed column (3 cm
in inside diameter and 25 cm in length), and a 4% acrylonitrile
aqueous solution or 2.6% methacrylonitrile aqueous solution was
continuously fed via the top of the column at a rate of 100 ml/hr
at 10.degree. C. and at 25.degree. C. (for comparison) to react the
times shown in Table 5. Ratios of produced acrylamide or
methacrylamide at respective reaction stages were determined to
obtain the results shown in Table 5.
Table 5 ______________________________________ Reaction Yield of
acrylamide Yield of methacrylamide Time (%) (%) (hr) 10.degree. C.
25.degree. C. 10.degree. C. 25.degree. C.
______________________________________ 10 100 100 100 100 20 100 32
100 100 30 100 0 100 100 40 100 0 100 4 50 100 0 100 0 100 100 0
100 0 150 100 0 100 0 200 100 0 100 0 250 100 0 100 0 300 100 0 100
0 ______________________________________
EXAMPLE 11
Table 6 given below comparatively shows the activity of intact
cells and of immobilized cells with respect to various
microorganisms having an acrylamide-producing ability.
Preparation of immobilized cells
4 parts of intact cells (water content: 75%), 0.45 part of
acrylamide, 0.05 part of N,N'-methylenebisacrylamide, and 4 parts
of physiological saline were mixed to prepare a uniform suspension.
To this suspension were added 0.5 part of a 5%
dimethylaminopropionitrile aqueous solution and 1 part of a 2.5%
potassium persulfate aqueous solution, and the system was
maintained as 10.degree. to 15.degree. C. for 30 minutes to
polymerize. Subsequently, the thus obtained cell-containing gels
were crushed and washed with physiological saline to obtain 10
parts of the immobilized cells.
Measurement of the acrylamide-producing ability
0.8 part of the intact cells or 2 parts of the immobilized cells
were diluted with a 0.05 M phosphate buffer (pH: 8.0) to make 100
parts. Then, 1 part of each of the thus diluted solution was mixed
with 1 part of a 0.05 M phosphate buffer (pH: 8.0) containing 2%
acrylonitrile and, after reacting at 10.degree. C. for 30 minutes
under stirring, acrylamide produced in the reaction solution was
determined to calculate the acrylamide-producing ability of each of
intact cells and immobilized cells.
Table 6 ______________________________________ Acrylamide-producing
ability* Microorganism Intact cells Immobilized cells
______________________________________ Strain N-771 genus
Corynebacterium 10.7 10.8 (FERM-P No. 4445) Strain N-774, genus
Corynebacterium 6.0 5.8 (FERM-P No. 4446) Strain N-775, genus
Nocardia 2.6 2.5 (FERM-P No. 4447)
______________________________________ *Amount of acrylamide (g)
produced by reacting for 1 hour per 1 g of dry bacterial cells.
EXAMPLE 12
40 parts of the washed cells of the strain N-771 (water content:
75%) prepared by aerobic culture using a culture medium (pH: 7.2)
containing 1% glucose, 0.5% peptone, 0.3% yeast extract and 0.3%
malt extract, 4.5 parts of acrylamide, 0.5 part of
N,N'-methylenebisacrylamide and 40 parts of physiological saline
were mixed to prepare a uniform suspension. To this suspension were
added 5 parts of a 5% dimethylaminopropionitrile aqueous solution
and 10 parts of a 2.5% potassium persulfate aqueous solution, and
maintained at 10.degree. C. for 30 minutes to polymerize. The thus
obtained massive, cell-containing gels were crushed into small
particles and washed well with physiological saline to obtain 100
parts of the immobilized cells.
5 jacketed columns, 3 cm inside diameter, 25 cm in length, each
filled with 40 g of the immobilized cells were connected to each
other in series, and a 4.5% acrylonitrile aqueous solution (using a
0.05 M phosphate buffer; pH: 8.0) was allowed to flow down via the
top of column No. 1 at 10.degree. C. at a flow-down rate of 50
ml/hr (SV.apprxeq.0.5 hr.sup.-1). Subsequently, 100 parts of the
eluate was mixed with 4.5 parts of acrylonitrile, and allowed to
flow down via the top of column No. 2 at a flow-down rate of 52.3
ml/hr (SV.apprxeq.0.53 hr.sup.-1). The eluate was then similarly
allowed to consecutively flow down through column No. 3 while
controlling the flow-down rate at 54.5 ml/hr (SV.apprxeq.0.54
hr.sup.-1), column No. 4 at 56.8 ml/hr (SV.apprxeq.0.57 hr.sup.-1),
and column No. 5 at 59 ml/hr (SV.apprxeq.0.59 hr.sup.-1) for 48
hours. Thus, there was continuously obtained an eluate at a
reaction ratio of 100%. Additionally, the acrylamide concentration
in this eluate was 25.5%.
EXAMPLE 13
7 jacketed columns, 3 cm inside diameter and 25 cm in length,
filled with 40 g of the immobilized cells prepared in the same
manner as in Example 12 were connected to each other in series, and
a 2.5% methacrylonitrile aqueous solution dissolved in a 0.05 M
phosphate buffer (pH: 8.0) was allowed to flow down through column
No. 1 via the top thereof at a flow-down rate of 100 ml/hr
(SV.apprxeq.1.00 hr.sup.-1). Then, 100 parts of the eluate was
mixed with 2.5 parts of methacrylonitrile and allowed to flow down
through column No. 2 via the top thereof at a flow-down rate of 103
ml/hr (SV.apprxeq.1.03 hr.sup.-1).
The eluate was then similarly allowed to consecutively flow down
through column No. 3 while controlling the flow-down rate at 105
ml/hr (SV.apprxeq.1.05 hr.sup.-1), column No. 4 at 108 ml/hr
(SV.apprxeq.1.08 hr.sup.-1), column No. 5 at 110 ml/hr
(SV.apprxeq.1.10 hr.sup.-1), column No. 6 at 113 ml/hr
(SV.apprxeq.1.13 hr.sup.-1), and column No. 7 at 115 ml/hr
(SV.apprxeq.1.15 hr.sup.-1) for 48 hours. The reaction ratio in
this eluate was 100%, and the concentration of methacrylamide in
the eluate was 19.3%.
EXAMPLE 14
45 parts of the washed cells of the strain N-775 (water content:
78%) prepared by aerobic culture using a culture medium containing
1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract,
0.1% acetonitrile, 0.1% KH.sub.2 PO.sub.4 and 0.05%
MgSO.sub.4.7H.sub.2 O, 4.5 parts of acrylamide, 0.5 part of
N,N'-methylenebisacrylamide and 40 parts of physiological saline
were mixed to prepare a uniform suspension. To this were added 5
parts of a 5% dimethylaminopropionitrile aqueous solution and 10
parts of a 2.5% potassium persulfate aqueous solution, and
maintained at 10.degree. C. for 30 minutes. The thus obtained
massive, cell-containing gels were crushed into small particles,
and washed well with physiological saline to obtain 100 parts of
the immobilized cells.
The immobilized cells were filled in a section column comprising
several sections each of which had a volume of 100 ml and contained
40 g immobilized cells. A 2.5% methacrylonitrile solution (using a
0.05 M phosphate buffer; pH: 8.0) was continuously fed at a rate of
100 ml/hr via the top of the uppermost section while maintaining
the temperature inside the column at 15.degree. C., and
methacrylonitrile at a rate of 3 ml/hr was added via the tops of
each of sections Nos. 2 to 7 for 48 hours to conduct the reaction.
In this case, no methacrylonitrile was detected in the eluate from
the bottom of the 7th section of the column. Thus, the reaction was
100%. The content of methacrylamide in this eluate was 19.3%.
EXAMPLE 15
40 parts of the washed cells of the strain CBS 717.73 (the strain
described in Examples of U.S. Pat. No. 4,001,081) obtained by
culturing in the same manner as in Example 12 (water content: 75%),
4.5 parts of acrylamide, 0.5 part of N,N'-methylenebisacrylamide
and 40 parts of physiological saline were mixed to obtain a uniform
suspension. To this were added 5 parts of a 5%
dimethylaminopropionitrile aqueous solution and 10 parts of a 2.5%
potassium persulfate aqueous solution, and the system was
maintained at 10.degree. C. for 30 minutes to polymerize. The thus
obtained massive, cell-containing gels were crushed into small
particles, and well washed with physiological saline to obtain 100
parts of immobilized cells.
5 jacketed columns, 3 cm inside diameter, 25 cm in length, each
filled with 40 g of the immobilized cells were connected to each
other in series, and a 4.5% acrylonitrile aqueous solution (using a
0.05 M phosphate buffer; pH: 8.0) was allowed to flow down through
column No. 1 via the top thereof at a temperature of 10.degree. C.
at a flow-down rate of 50 ml/hr (SV=0.5 hr.sup.-1). Subsequently,
100 parts of the eluate was mixed with 4.5 parts of acrylonitrile,
and allowed to flow down through column No. 2 via the top thereof
at a flow-down rate of 52.3 ml/hr (SV.apprxeq.0.53 hr.sup.-1).
The eluate was then similarly allowed to consecutively flow down
through column No. 3 while controlling flow-down rate at 54.5 ml/hr
(SV.apprxeq.0.54 hr.sup.-1), column No. 4 at 56.8 ml/hr
(SV.apprxeq.0.57 hr.sup.-1), and column No. 5 at 59 ml/hr
(SV.apprxeq.0.59 hr.sup.-1). Analysis of the eluate from column No.
5 48 hours after the initiation of the flowing down of the solution
revealed an existence of a slight amount of acrylonitrile, and the
concentration of acrylamide was determined to be 24.8%.
While the invention has been described in detail and with reference
to specific embodiments thereof, it will be apparent to one skilled
in the art that various changes and modifications can be made
therein without departing from the spirit and scope thereof.
* * * * *